(Hypertension. 1996;27:975-978.)
© 1996 American Heart Association, Inc.
Articles |
Genetic Analysis of Renin Gene Expression in Rat Adrenal Gland
From the First Department of Internal Medicine, Shiga (Japan) University of Medical Sciences.
Correspondence to Naoharu Iwai, MD, First Department of Internal Medicine, Shiga University of Medical Sciences, Tsukinowa Seta, Ohtsu-shi, Shiga-ken, Japan.
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Abstract |
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Abstract We examined the mechanism of the increased renin mRNA concentration in the adrenal glands of spontaneously hypertensive rats (SHR). In 52 female F2 rats (25 to 27 weeks of age) derived from SHR and Wistar-Kyoto rats, we determined blood pressure, renin mRNA concentration in the adrenal gland, plasma renin activity, plasma aldosterone concentration, and genotype of the renin gene. Eighteen of the F2 rats were fed a high salt (8%) diet for 14 days. The renin mRNA concentration in the adrenal glands showed a significant correlation with the genotype of the renin gene in the normal salt diet group (P<.0001), whereas this relationship was not observed in the high salt diet group. Multivariate analysis revealed that the plasma aldosterone concentration in the normal diet group was significantly explained (P=.0004, R2=.454) by plasma renin activity (P=.0005), the renin mRNA concentration in the adrenal gland (P=.0496), and the genotype of the renin gene (P=.0236). The SHR allele of the renin gene was associated with a lower aldosterone concentration. On the other hand, in the high salt diet group, only the genotype of the renin gene showed a significant relationship with plasma aldosterone concentration (P=.0237). Again, the SHR allele of the renin gene was associated with a lower aldosterone concentration. We can conclude that the higher renin mRNA concentration in the SHR adrenal glands is governed by the SHR allele of the renin gene or renin gene locus. The renin mRNA concentration in the adrenal gland exerts a minor influence on aldosterone synthesis. Paradoxically, the SHR allele of the renin gene or renin gene locus confers a lower rate of aldosterone synthesis at 25 to 27 weeks of age, the mechanism of which remains to be determined.
Key Words: renin • genes • aldosterone • adrenal glands
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Introduction |
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In a previous article,1 we reported that the renin mRNA concentration in the adrenal gland and various parts of the central nervous system was higher in SHR than in age-matched WKY. The increased renin mRNA concentration in these extrarenal tissues may be involved in the pathogenesis or maintenance of hypertension in SHR. The increased renin mRNA concentration in these extrarenal tissues may result from the increased BP in SHR, genetic differences in the renin gene, or genetic differences in other genes influencing renin gene expression and may be responsible for the pathogenesis or maintenance of hypertension.
In Dahl salt-sensitive and salt-resistant rats, a genetic analysis indicated that the lower expression of renin in the adrenal gland of the salt-sensitive rats was not caused by volume expansion and the higher expression of renin in the adrenal gland of the salt-resistant rats was a polygenic autosomal dominant trait.2
In the present study, we examined the mechanisms of increased renin mRNA concentration in the adrenal glands of SHR. In F2 rats derived from SHR and WKY, we assessed various phenotypic parameters, including BP, renin mRNA concentration in the adrenal glands, PRA, and plasma aldosterone concentration and determined the renin gene genotype in these rats. We also addressed the possible physiological roles of the renin expressed in the adrenal gland.
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Methods |
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Animals
Inbred male SHR and female WKY were obtained from Charles River Laboratories (Atsugi, Japan). From these, we raised 52 female F2 rats for the present study. The rats were housed in a temperature-controlled room with lights on from 6 AM to 6 PM and were fed normal pelleted rat chow (0.39% NaCl) and tap water ad libitum. BP was measured on three separate occasions between 9 and 11 AM by the tail-cuff method when the rats were between 25 and 27 weeks of age. The mean BP of the three measurements was used as the BP value for each rat. The rats were killed by decapitation, and blood was collected into prechilled tubes containing EDTA disodium salt (2 mg/mL) for PRA and plasma aldosterone concentration. The plasma was separated by centrifugation at 3000 rpm for 15 minutes at 4°C and stored at -80°C until use. PRA, measured in terms of the rate of Ang I generation, was determined with radioimmunoassay kits (Dainapot Co Ltd), as was plasma aldosterone concentration (Diagnostic Products Corp).
At 25 weeks of age, 18 of the rats began a high salt diet containing 8% NaCl for 14 days. BP was measured on two separate occasions between days 12 and 14 of the high salt diet. The rats were killed by decapitation on day 15 of the high salt diet, and blood was collected as described above for biochemical analysis.
All animal protocols were approved by the University Animal Use and Care Committee.
DNA and RNA Analyses
RNA was isolated as described previously.3 The
quality of the isolated RNA was confirmed by ethidium bromide staining.
The expression level of renin mRNA was determined by RT-PCR because of
the relatively low expression levels of renin mRNA in the adrenal
gland, as described previously.1 The validity of this
method has been discussed in detail.1
Briefly, 4-µg samples of total RNA mixed with a known amount of
deletion-mutated renin cRNA (0.4x106 molecules
for high salt, 1.6x106 for normal salt) were
reverse transcribed with random primers as a primer. The resulting cDNA
mixture was purified by phenol/chloroform extraction and two rounds of
ethanol precipitation with ammonium acetate and was dissolved in 40 mL
H2O. Five milliliters of the cDNA mixture was amplified in
a total volume of 25 mL of a reaction mixture containing 50 mmol/L KCl,
10 mmol/L Tris-HCl (pH 8.3), 2.0 mmol/L MgCl2,
0.01% (wt/vol) gelatin, 0.2 mmol/L dNTP, 50 nmol/L
[
-32P]dCTP (3000 Ci/mmol), 25 pmol of primers 1 and 2,
and 0.5 U of Taq DNA polymerase (Toyobo). The PCR
amplification profile included an initial denaturation step at 94°C
for 1 minute followed by 35 cycles at 94°C for 1 minute, 58°C for 1
minute, and 74°C for 2 minutes. PCR products were electrophoresed
on a 1.7% agarose gel for visual inspection and on a 5%
polyacrylamide gel for precise quantification, as described
previously.1 The sense primer 1 was
5'-CTGGGAGGCAGTGACCCTCAACATTACCAG-3' (747-776), and the
antisense primer 2 was 5'-GAGAGCCAGTATGCACAGGTCATCGTTCCT-3'
(1118-1089).
In the present study, the renin mRNA concentration in samples was calculated as follows: Expression Level (molecules per microgram)=(Amount of Mixed Deletion-Mutated cRNA)/4x(Intensity of 372-bp Fragment/Intensity of 263-bp Fragment)x0.625, where 0.625 is the ratio of the dCTP content of the 263-bp fragment to that of the 372-bp fragment.
Genomic DNA was isolated from the liver according to a previously described method.4 Renin genotype was determined by the PCR method, in which the presence (SHR allele) or absence (WKY allele) of the HindIII site in intron 5 was detected. The sense primer 1 was 5'-CTGTCTTCGACCACATTCTCTCCCAGAC-3' (exon 4) and the antisense primer 2 was 5'-GAGGGTCACTGCCTCCCAGCACCACTTC-3' (exon 5). The renin primer sequences were based on a previous report.5 Genomic DNA (50 ng) was amplified in 25 mL of reaction mixture containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 2.0 mmol/L MgCl2, 0.01% (wt/vol) gelatin, 0.2 mmol/L dNTP, 25 pmol of primers 1 and 2, and 10 U of Taq DNA polymerase. The PCR amplification profile included an initial denaturation step at 94°C for 1 minute followed by 32 cycles at 94°C for 1 minute, 58°C for 1 minute, and 74°C for 4 minutes. The PCR product was digested with an excess quantity of restriction enzyme HindIII and resolved on a 0.8% agarose gel.
Statistical Analysis
Statistical analysis was performed by both one- and
two-way ANOVA and multiple regression analyses.
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Results |
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Characteristics of F2 Rats
Table 1
shows the characteristics of the 34
F2 rats fed a normal salt diet. The genotype of the
renin gene had no significant effects on BP. Figs 1 and 2 show typical analyses of renin
mRNA concentration in adrenal glands and of renin gene genotyping. As
summarized in Table 1, renin mRNA concentration in the adrenal glands
showed a significant correlation with the genotype of the renin
gene (P<.0001).
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Table 2 shows the characteristics of the 18
F2 rats fed a high salt diet. The high salt diet
significantly increased BP (P=.0024) and significantly
reduced PRA (P=.0024), renin mRNA concentration in the
adrenal glands (P<.0001), and plasma
aldosterone (P<.0001) (P values were
calculated by two-way ANOVA; x1=type of diet,
x2=genotype of the renin gene). No significant
correlation was observed between the genotype of the renin gene
and the renin mRNA concentration in the adrenal glands of rats fed a
high salt diet. Again, the genotype of the renin gene had no
significant effects on BP (Table 2). A significant correlation
(P=.0237) between the genotype of the renin gene and
plasma aldosterone concentration was observed in the high
salt diet group (Table 2). The SHR allele of the renin gene was
associated with a lower aldosterone concentration than was
the WKY allele of the renin gene.
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Aldosterone and Adrenal Renin
The adrenal renin-angiotensin system has been
suggested to be involved in aldosterone
synthesis.6 Therefore, we investigated the relationship
between plasma aldosterone and the renin mRNA concentration
in the adrenal glands. Multiple regression analysis revealed
that the plasma aldosterone concentration was explained by
PRA, renin mRNA concentration in the adrenal glands, and the
genotype of the renin gene (Table 3), for which
the WW, SW, and SS genotypes were assigned 1, 2, and 3,
respectively (W indicating the WKY allele, and S, the SHR
allele). In the high salt diet group, only the genotype of
the renin gene was a predictor of plasma aldosterone
concentration (Table 2
). No correlation was observed between plasma
aldosterone concentration and either the renin mRNA
concentration in the adrenal glands or PRA in the high salt diet
group.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The most important finding in the present study is that the SHR allele of the renin gene showed a significant correlation with a higher renin mRNA concentration in the adrenal glands of rats fed a normal salt diet. However, this correlation was not observed in rats fed a high salt diet. In the high salt diet group, the SHR allele of the renin gene showed a significant correlation with a lower aldosterone concentration. This was also revealed by multiple regression analysis in the normal salt diet group. The renin gene locus showed no significant influence on BP in the present F2 rat population.
Adrenal Renin and Genotype of the Renin Gene
Although the genotype of the renin gene strongly
influenced the renin mRNA concentration in the adrenal glands of rats
fed a normal salt diet, the genotype showed no significant
influence on PRA or the renin mRNA concentration in the adrenal glands
of rats fed a high salt diet.
The SHR allele of the renin gene also showed a significant correlation with a higher renin mRNA concentration in the brain stem of rats in each diet group (data not shown). The lack of a relationship between the SHR allele of the renin gene and PRA may be the result of a compensatory mechanism that suppresses renin gene expression. Higher renin expression in the kidneys might lead to extracellular volume expansion, which in turn might suppress renin gene expression in the kidneys.
The expression of the renin gene in the adrenal glands is regulated by dietary salt content.1 However, the factors that modulate renin gene expression in the adrenal glands have yet to be fully clarified. The reactivity to some of these factors may be enhanced in the presence of the SHR allele of the renin gene. Bilateral nephrectomy is the most potent upregulator of renin gene expression in the adrenal glands. We have analyzed the renin mRNA concentrations in the adrenal glands of male F2 rats at 48 hours after bilateral nephrectomy. The renin mRNA concentration had significantly increased after bilateral nephrectomy, and the SHR allele of the renin gene was found to correlate significantly with a higher renin mRNA concentration (data not shown).
Physiological Function of Adrenal
Renin
The significant correlation of the SHR allele of the renin
gene with a lower plasma aldosterone concentration and the
significant positive correlation of the renin mRNA concentration in the
adrenals with the plasma aldosterone concentration was
puzzling because the SHR allele of the renin gene is associated
with a higher renin mRNA concentration in the adrenal glands. Our
results indicate that aldosterone synthesis is responsive
to the renin mRNA concentration in the adrenal glands and that the
lower responsiveness of aldosterone synthesis to Ang II may
be associated with the SHR allele of the renin gene. In the normal
salt diet group, PRA was the strongest determinant of plasma
aldosterone concentration, whereas the influences of the
renin mRNA concentration in the adrenal glands and that of the
genotype of the renin gene were minor. Conversely, in the high
salt diet group, PRA was found to be no longer a determinant of plasma
aldosterone concentration because the PRA level was very
low. The renin mRNA concentration in the adrenal glands was also lower
in the high salt diet group and was found not to be a determinant of
aldosterone concentration. Only the genotype of the
renin gene was found to be a determinant of plasma
aldosterone, in that the SHR allele of the renin gene
was associated with a lower plasma aldosterone
concentration (Table 2).
Three possible explanations of the association between the SHR allele of the renin gene and a lower plasma aldosterone concentration must be considered. One possibility is that the SHR allele of the renin gene is primarily involved in the enhancement of renin gene expression in the adrenal glands, which leads to a higher Ang II level in the adrenal glands. The higher Ang II level might lead to downregulation of the Ang II receptor and/or other components involved in aldosterone synthesis. Administration of a high salt diet for only 14 days might not be sufficient to restore the expression levels of these components.
The second possibility is that the renin gene locus is closely linked to a gene that influences aldosterone synthesis and that the SHR allele of the renin gene is linked to a genotype of a gene that confers low aldosterone synthesis. The association of the SHR allele of the renin gene with a higher expression of renin mRNA in the adrenal glands may be a manner of compensating for the reduction in aldosterone synthesis.
Another possibility is that renin mRNA translation or renin protein posttranslational processing may be impeded in the adrenal glands of SHR and mature active renin is thereby suppressed. Lower levels of mature active renin in the adrenal glands of SHR might lead to lower aldosterone synthesis, which in turn might increase expression of the renin gene in the adrenal glands. According to this hypothesis, the genomic locus very close to the renin gene locus should be responsible for renin mRNA translation or renin posttranslational processing because the SHR allele of the renin gene is associated with higher renin mRNA concentration in the adrenals. Thus, this third possibility may be intrinsic to the second one. However, the elevated renin mRNA concentration in SHR appears to correlate with elevated renin protein levels. Indeed, markedly elevated active renin protein levels in the adrenal glands have been reported in both SHR7 and stroke-prone SHR.8 Elevated active renin protein levels have also been reported in various parts of the SHR central nervous system.9 These reports appear to contradict the hypothesis that renin mRNA translation or renin protein posttranslational processing may be impeded in the adrenals of SHR.
The transgenic TGR(mREN-2)27 rat is a new monogenic model of hypertension, with high expression of the additional mouse Ren-2d gene in extrarenal tissues, especially in the adrenal cortex.10 This model may be useful for interpreting our present results. In this rat model, urinary excretion of deoxycorticosterone, corticosterone, 18-hydroxycorticosterone, and aldosterone has been reported to be elevated during the development of hypertension.11 Despite persistently elevated mouse Ren-2d gene expression in the adrenal cortex, urinary excretion of these steroids returns to normal by 30 weeks of age, suggesting that the high Ang II level in the adrenals may lead to downregulation of some components of aldosterone synthesis, in turn leading to reduced responsiveness of aldosterone synthesis to Ang II. This progression may also apply to the F2 rats analyzed in the present study. Analysis of younger F2 rats will be necessary for clarification.
In summary, the present study demonstrates that the higher renin mRNA concentration in SHR adrenals is due to the SHR allele of the renin gene or renin gene locus. The renin mRNA concentration in the adrenals exerts only a minor influence on plasma aldosterone concentration. Paradoxically, the SHR allele of the renin gene or renin gene locus confers a lower aldosterone concentration in F2 rats of 25 to 27 weeks of age. The mechanisms of this phenomenon remain to be determined.
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Selected Abbreviations and Acronyms |
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Received September 22, 1995; first decision October 16, 1995; accepted December 21, 1995.
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References |
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- Iwai N, Inagami T, Ohmichi N, Nakamura Y, Saeki Y, Kinoshita M. Differential regulation of rat AT1a and AT1b receptor mRNA. Biochem Biophys Res Commun. 1992;188:298-303. [Medline] [Order article via Infotrieve]
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[Abstract/Free Full Text] - Fukamizu A, Nishi K, Cho T, Saito M, Nakayama K, Ohkubo H, Nakanishi S, Murakami K. Structure of the rat renin gene. J Mol Biol. 1988;201:443-450. [Medline] [Order article via Infotrieve]
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Naruse M, Inagami T. Markedly elevated specific
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[Abstract/Free Full Text] - Schelling P, Meyer D, Loos HE, Speck G, Phillips MI, Johnson AK, Ganten D. A micromethod for the measurement of renin in the brain nuclei, and its application in spontaneously hypertensive rats. Neuropharmacology. 1982;21:455-463. [Medline] [Order article via Infotrieve]
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